Reis et al BMC Genomics (2019) 20:885 https://doi.org/10.1186/s12864-019-6206-z RESEARCH ARTICLE Open Access Comparative genomics reveals a novel genetic organization of the sad cluster in the sulfonamide-degrader ‘Candidatus Leucobacter sulfamidivorax’ strain GP Ana C Reis1,2, Boris A Kolvenbach2, Mohamed Chami3, Luís Gales4,5,6, Conceiỗóo Egas7,8, Philippe F.-X Corvini2 and Olga C Nunes1* Abstract Background: Microbial communities recurrently establish metabolic associations resulting in increased fitness and ability to perform complex tasks, such as xenobiotic degradation In a previous study, we have described a sulfonamidedegrading consortium consisting of a novel low-abundant actinobacterium, named strain GP, and Achromobacter denitrificans PR1 However, we found that strain GP was unable to grow independently and could not be further purified Results: Previous studies suggested that strain GP might represent a new putative species within the Leucobacter genus (16S rRNA gene similarity < 97%) In this study, we found that average nucleotide identity (ANI) with other Leucobacter spp ranged between 76.8 and 82.1%, further corroborating the affiliation of strain GP to a new provisional species The average amino acid identity (AAI) and percentage of conserved genes (POCP) values were near the lower edge of the genus delimitation thresholds (65 and 55%, respectively) Phylogenetic analysis of core genes between strain GP and Leucobacter spp corroborated these findings Comparative genomic analysis indicates that strain GP may have lost genes related to tetrapyrrole biosynthesis and thiol transporters, both crucial for the correct assembly of cytochromes and aerobic growth However, supplying exogenous heme and catalase was insufficient to abolish the dependent phenotype The actinobacterium harbors at least two copies of a novel genetic element containing a sulfonamide monooxygenase (sadA) flanked by a single IS1380 family transposase Additionally, two homologs of sadB (4-aminophenol monooxygenase) were identified in the metagenome-assembled draft genome of strain GP, but these were not located in the vicinity of sadA nor of mobile or integrative elements Conclusions: Comparative genomics of the genus Leucobacter suggested the absence of some genes encoding for important metabolic traits in strain GP Nevertheless, although media and culture conditions were tailored to supply its potential metabolic needs, these conditions were insufficient to isolate the PR1-dependent actinobacterium further This study gives important insights regarding strain GP metabolism; however, gene expression and functional studies are necessary to characterize and further isolate strain GP Based on our data, we propose to classify strain GP in a provisional new species within the genus Leucobacter, ‘Candidatus Leucobacter sulfamidivorax‘ Keywords: Sulfonamides, Bacterial consortium, Phylogenetic analysis, Metagenome-assembled genome, Cryo-transmission electron microscopy * Correspondence: opnunes@fe.up.pt Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering - LEPABE, Department of Chemical Engineering, University of Porto, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal Full list of author information is available at the end of the article © The Author(s) 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Reis et al BMC Genomics (2019) 20:885 Background Microbial communities are known to establish sophisticated metabolic interactions in order to achieve complex and energy-expensive tasks [1–5] These syntrophic relationships are frequently studied in bacterial pathogens and symbiotic bacteria, where the interaction with the host often drives progressive adaptation, mutation, and subsequently, gene loss These phenomena may render the bacteria “unculturable” or difficult to grow under standard laboratory conditions [6–11] On the contrary, the phenomena underlying metabolic cooperation and competition within environmental communities are often more complex, and their implications for microbial ecology are still poorly understood [5, 11] These communities recurrently exchange metabolites or co-factors and are often associated with xenobiotic-degraders thriving in polluted environments [5, 11–15] This syntrophy has been previously observed in terephthalate-degrading communities [1, 2], in anammox-associated communities [3, 4, 16], in the dichloromethane-degrader ‘Candidatus Dichloromethanomonas elyunquensis’ [17], and in members of the candidate phylum ‘Candidatus Latescibacteria’, that thrives in hydrocarbon-impacted environments [18, 19] However, to date, no representatives of these groups could be isolated as pure cultures, and their metabolic needs are difficult to assess Terephthalatedegraders, for instance, thrive in an intricate network formed between H2-producing syntrophs and methanogenic archaea, with numerous other secondary interactions essential for the stability of the consortium [1, 2] Anammox bacteria were shown to form stable biofilm communities with ammonia-oxidizing bacteria (AOB), that appear to be essential to protect the sensitive anammox species from atmospheric O2 [3, 4, 20, 21] The evolution of these communities is driven by selective pressure and stress and may result in complex syntrophic relationships that may lead to niche-specialization and dependency on other members of the community In order to characterize the members of these communities, cellsorting and metagenomics approaches are being used to circumvent the need for cultivation [15] Furthermore, these studies are frequently complemented with comparative genomics which has emerged as a valuable tool to determine the evolution and functional prediction between even distantly related bacteria [14, 22, 23] The cultivation of several members of the ubiquitous SAR11 aquatic bacteria, with no closely related culturable relatives, has been made possible by in silico metabolic studies and nextgeneration sequencing approaches [24] Furthermore, the evolution of this abundant group of Alphaproteobacteria and their ecological importance has been further elucidated using comparative genomic approaches [25] In a previous study, we have described a microbial consortium between Achromobacter denitrificans strain PR1 and strain Page of 23 GP that depends on strain PR1’s presence for growth [26] Strain GP showed the highest pairwise similarity of its 16S rRNA gene sequence to members of the genus Leucobacter Independently of the tested culture media, cofactors and culture conditions no pure cultures were obtained for strain GP [26] To characterize strain GP, we have sequenced the two-member consortium and reconstructed its draft genome Also, we have performed comparative genomic studies in order to understand its phylogenetic relationship with other members of the Leucobacter genus and propose the hypothesis that may allow us to understand why this strain has eluded isolation in previous studies Results and discussion Morphological and physiological characterization of the consortium The microbial consortium between strain A denitrificans and the low-abundant strain GP was visualized by Cryo-TEM during mid-stationary phase (Fig 1a and b), as well as by FISH (see Additional file Figure S1) As expected, strain PR1 showed the typical morphology of Gram-negative rods with an average cell size of 801.3 ± 40.2 nm (width), 1332 ± 98.7 nm (length) and 38.2 ± 6.5 nm (periplasmic space) (Fig 1a) Moreover, peritrichous flagella were observed by negative stain electron microscopy (FG, see Additional file Figure S2) Although flagella have not been previously reported for the type strain of A denitrificans, their presence has been repeatedly observed in other strains from this species [27] and other species of the Achromobacter genus [28, 29] Conversely, strain GP displayed the typical morphology of Gram-positive rods Its cells showed an average size of 506.6 ± 30.1 nm (width) and 1341.0 ± 29.7 nm (length) (Fig 1b), and the rigid cell wall of this organism had an average thickness of 20.6 ± 2.2 nm No flagella were observed for this bacterium, suggesting that it is nonmotile, like previously reported for other members of the Leucobacter genus [30, 31] The two members of the consortium revealed significant differences regarding their respective tolerances toward temperature, pH and salinity (Fig 2) While the abundance of strain PR1 was constant when incubating at 22, 30 and 37 °C, respectively, strain GP abundance was significantly reduced at 37 °C (p < 0.05) when compared to the other tested temperatures Strain GP also showed a lower abundance when incubated at pH 5.5, in comparison to cultures incubated in media at neutral (pH 7.2) and basic (pH 9.5) pH values (Fig 2) As it is typically observed for members of the Achromobacter genus [32], NaCl concentrations up to 4% (w/ v) did not influence the abundance of strain PR1; however, its abundance was significantly reduced above this value (Fig 2) Although the absolute Reis et al BMC Genomics (2019) 20:885 Page of 23 Fig Electron micrographs of frozen hydrated Achromobacter denitrificans strain PR1 (a) and strain GP (b) PM – Plasma membrane; OM – Outer membrane; FG – Flagellum; CW – Cell wall; C – Carbon support grid amount of strain GP 16S rRNA copy numbers also decreased above 4% NaCl (w/v), the relative abundance of this strain in the consortium was significantly higher (ranging from 0.24% at 0% NaCl, to a maximum of 4.26% at 8% NaCl) Interestingly, the abundance of strain GP was significantly lower in complex media (Tryptic Soy Broth, TSA; Brain-Heart Infusion, BHI; and Reasoner’s 2A medium, R2A) than in mineral media with succinate and trace amounts of yeast extract (MMSY, Fig 2) These results suggest that strain GP is possibly oligotrophic, unlike previously described for members of the Leucobacter genus, which thrive in complex media, such as BHI enriched with peptone and yeast extract, as observed for L luti RF6T [30] Analysis of the metagenome-assembled genome of strain GP The analysis of the metagenomic contigs with SSU finder (rRNA small subunit) from CheckM [34] revealed the presence of only two phylogenetic distinct organisms: one identified as A denitrificans PR1 and the other as strain GP The reconstruction of strain GP’s genome from whole-consortium sequencing generated a metagenomeassembled genome (MAG) consisting of 11 contigs, with 3.84 Mb, 3621 coding sequences (CDS), 69.68% in G + C Fig Abundance of strain PR1 and strain GP after 15 h incubation at different pH, salinity (in DLB), temperatures (in MMSY) and media (R2A, TSA, BHI and MMSY) The values for copies of the 16S rRNA gene per ml are plotted in logarithmic scale Values are the mean values of triplicates and the error bars represent the standard deviation Significant differences in strain GP abundance are indicated by a, b, c and d (from higher to lower values of the mean) as determined by two-way ANOVA (pH, temperature and salinity) or one-way ANOVA (PR1/GP ratio in R2A, TSA, BHI and MMSY) and the Tukey test at p < 0.05 within each tested condition [33] 95.87 95.87 95.94 L musarum subsp japonicus CBX130T L musarum subsp musarum CBX152T L salsicius M1-8T L zeae CC-MF41T L triazinivorans JW-1 95.88 96.4 96.52 T 96.81 96.23 L chromiiresistens J31T L massiliensis 122RC15T 96.15 L chromiireducens subsp solipictus TAN 31504T L luti RF6T 96.23 L chromiireducens subsp chromiireducens L-1T 96.37 95.43 L chironomi DSM 19883T 96.79 QYAC00000000 95.87 L celer subsp astrifaciens CBX151T L komagatae DSM 8803T QYAD00000000 96.23 L aridicollis L-9T L chromiiresistens NS354 QZLF00000000 – Candidatus Leucobacter sulfamidivorax’ GP QYAB00000000 GCA_004208635.1 GCA_000350525.1 GCA_001273845.1 GCA_001273855.1 GCA_002982315.1 QYAG00000000 GCA_006716085.1 GCA_001477055.1 GCA_000231305.1 GCA_000421845.1 GCA_001273835.1 QYAE00000000 Genome*/ assembly accession no 16S rRNA pairwise similarity to GP (%) Strain 99.85 100.00 99.42 99.85 99.56 100.00 100.00 98.54 95.03 100.00 99.42 100.00 100.00 98.83 99.27 95.91 Completeness (%) 0.58 0.88 0.00 0.58 0.58 1.75 0.58 1.75 0.00 0.00 2.05 0.58 0.88 0.00 0.58 0.58 Contamination (%) 28 125 144 24 194 11 27 235 11 Number contigs/ scaffolds 3.47 3.48 3.18 3.44 3.59 3.14 3.62 3.75 2.79 3.21 3.54 3.22 2.96 4.14 3.56 3.84 Genome size (Mb) 69.37 70.6 – 2,226,772 64.5 66.8 66.8 71.0 69.4 66.6 70.8 70.3 68.9 67.0 69.9 69.1 67.3 69.68 G + C content (%) 197,637 200,785 248,155 232,656 1,858,864 3,292,530 35,220 2,823,343 451,461 623,960 268,438 349,813 888,847 956,104 Contig N50 3042 2978 2741 3147 3311 2789 3088 3253 2423 2895 3096 2843 2662 3661 3212 3621 Number of CDS This study [145] [65] [144] [144] [143] This study Unpublished [142] [31] This study This study [64] [122] This study This study Reference Table List of named species of the Leucobacter genus used in the phylogenetic and comparative studies Assembly quality was calculated using QUAST [49] with a minimum contig size set to 200 bp Completeness and contamination were computed with CheckM [34] 16S rRNA pairwise similarity was computed with the global alignment tool in the EzBioCloud web server [126] Reis et al BMC Genomics (2019) 20:885 Page of 23 Reis et al BMC Genomics (2019) 20:885 and a total mapped coverage of 61x (Table 1) In spite of an enrichment step with 2-phenylethanol, only 18.5% of the total of reads obtained with Oxford Nanopore (ONT) and Illumina technologies were mapped to strain’s GP MAG, while the remaining reads mapped to the complete genome of A denitrificans PR1 (148x coverage in the consortium, see Additional file Table S1), previously determined [35] The MAG of strain GP encoded a complete rRNA operon and harbored two copies of the 5S and one copy of the 16S and 23S rRNA subunits, respectively Moreover, analysis with tRNAscan-SE [36] identified 44 tRNA encoding for all 20 amino acids CheckM [34] analysis showed high completeness and low contamination values for this assembly, as only marker genes were not detected in the draft genome and markers had copies in the assembly (95.9% completeness and 0.6% contamination, respectively, see Additional file Table S2) Therefore, according to Bowers et al [37], these findings indicate that this methodology allowed the reconstruction of a high-quality MAG for strain GP (Table 1) Analysis of mobile and conjugative elements The identification of potential plasmids and other mobilizable elements in the genome of strain GP was performed in silico by measuring differences in coverage and G + C content between the contigs of the draft assembly Compared to the average values for all contigs, at least three contigs (5, and 9) showed a significantly higher coverage, and lower G + C content (see Additional file Table S1) The differential coverage among contigs was observed consistently with both Illumina and ONT libraries, which were prepared from different biological replicates of the consortium Therefore, these differences are unlikely to arise from library preparation and sequencing bias The differences encountered suggest that these contigs may represent potential plasmids with an average copy number per cell of approximately 2–3 (contigs and 9) and (contig 7), respectively Furthermore, conserved domain search and CONJscan revealed the presence of several elements linked to plasmid replication, stability, partitioning, conjugation, and mobility (Table 2) Out of these three contigs, only contig (11.8 kb) was marked as circular by Circlator [38]; however, it had no relevant hits to other plasmids available in the National Center for Biotechnology Information (NCBI) database Contrarily, contig (22.5 kb) featured residual homology to a new plasmid found in Cnuibacter physcomitrellae XAT (accession number CP020716.1, 4285 bp alignment with 99% identity to this plasmid), and the plasmid pKpn-35963cz from Klebsiella pneumoniae Kpn-35963cz (accession number MG252894.1, 2030 bp alignment with 99% identity to this plasmid) The respective homologous regions contained genes encoding for transposases and mercury resistance Both contigs and carry a gene encoding for Page of 23 a putative relaxase (locus tag: D3X82_18105, D3X82_ 18250, respectively) with a TrwC family domain (accession no pfam08751; E-value: 3.7e-28 and 7.6e-25, respectively), commonly observed in proteins from the MOBF (mobility) family (e.g., TraA from Arthrobacter sp Chr15, accession no ABR67091.1 [39]) This classification was further confirmed by CONJscan [40–42], which found that both D3X82_18105 (contig 7) and D3X82_18250 (contig 9) possess a highly conserved MOBF domain (Evalues of 5.3e-105 and 4.1e-106, respectively) Additional mobility elements were only found in contig This contig was found to harbor a putative plasmid replication protein (locus tag: D3X82_18090; Family: RepA_C; accession no: pfam04796; E-value: 9.0e-07) In this way, according to Guglielmini et al [42] and Smillie et al [43], the presence of a MOB element in contigs and suggests these putative elements are mobilizable but non-conjugative Contig 5, with 74 kb, was found to contain various integrative and conjugative elements (Table 2) [44] Besides, this contig contained all antimicrobial resistance genes found in the genome of strain GP (sul1, tet(33), aadA1, qacE), as well as two copies of the sadA gene encoding for the previously described sulfonamide monooxygenase [26] Table Homology searches for contig against the NCBI database [45] revealed residual homology to Enterobacter cloacae strain EclC2185’s genomic island (accession number MH545561.1, 5187 bp alignment with 99% identity to the genomic island of this strain) containing a class I integron with multi-drug resistance genes (aadA1, sul1, and qacE) Other significant alignments included regions conferring mercury resistance (Cnuibacter physcomitrellae XAT plasmid, accession number CP020716.1, 5928 bp alignment with 99% identity to this plasmid) and intergenic regions of the new plasmid pOAD2 from Flavobacterium sp KI723TI (accession number D26094.1, 14,820 bp alignment with 94% to this plasmid) According to conserved domain search and CONJscan analyses, two putative MOB elements were found in contig 5: (i) D3X82_17470, a relaxase from the MOBF family with a TwrC conserved domain (CONJscan domain search: E-value 1.3e-85); (ii) D3X82_17405, a relaxase from the MOBP1 family (CONJscan domain search: E-value 4.2e-40) Other essential mobilizable elements detected include a type IV coupling protein (T4CP, locus tag D3X82_17390) with a conserved VirD4 domain (CONJscan domain search: E-value 5.7e40) and a type IV secretion protein (T4SS, locus tag D3X82_17385) with a VirB4 domain (CONJscan domain search: E-value 1.4e-25) According to Smillie et al [43], these three elements (T4SS, T4CP and relaxases), represented in four locus tags in strain GP, are at the core of plasmid conjugation, however, no other known accessory proteins were detected in our analysis, presumably due to incomplete assembly and/or low identity to previously characterized proteins from the mating-pair formation Multi-drug and heavy metal resistance Mobilization and conjugative elements Replication, stabilization and partitioning System D3X82_17485 D3X82_17505 D3X82_17510 D3X82_17515 D3X82_17685 5 5 D3X82_18250 D3X82_18105 D3X82_17365 D3X82_17695 D3X82_17405 5 D3X82_17385 D3X82_17390 5 D3X82_17470 D3X82_18090 D3X82_17555 D3X82_17410 D3X82_17550 5 D3X82_17420 Locus tag Contig Mercury(II) reductase Sulfonamide-resistant dihydropteroate synthase Sul1 Quaternary ammonium compound efflux SMR transporter QacE delta ANT(3″)-Ia family aminoglycoside nucleotidyltransferase AadA1 Tet(A)/Tet(B)/Tet(C) family tetracycline efflux MFS transporter Sulfonamide monooxygenase SadA Conjugal transfer protein Conjugal transfer protein Relaxase Coupling protein (T4CP) Type IV secretion protein (T4SS) Conjugal transfer protein Plasmid mobilization relaxosome protein MobC Plasmid replication protein Single-stranded DNA-binding protein Plasmid partition protein A Toxin protein from a toxin/antitoxin system Description MerA Pterin_bind Multi_Drug_Res PRK13746 DUF4111 MFS_TetA NcnH CaiA Acyl-CoA_dh_2 TrwC MOBF TrwC MOBF Relaxase MOBP1 VirD4 T4CP2 VirB4 TrwC MOBF MobC RepA_C SSB_OBF BcsQ partition_RepA Zeta_toxin Family 7.6e-25 4.1e-106 pfam08751 n.a TIGR02053 pfam00809 pfam00893 PRK13746 pfam13427 cd17388 1.2e-171 1.8e-87 1.75e-28 0e+ 00 1.31e-41 2.4e-145 1.52e-84 1.93e-30 5.8e-20 3.7e-28 5.3e-105 pfam08751 n.a cd01159 COG1960 pfam08028 9.8e-10 4.20e-40 4.2e-16 5.7e-40 1.40e-25 9.0e-97 1.3e-85 5.7e-05 9.0e-07 8.5e-12 2.2e-43 5.7e-16 1.6e-19 E-value pfam03432 n.a COG3505 n.a n.a pfam08751 n.a pfam05713 pfam04796 cd04496 COG1192 TIGR03453 pfam06414 Accession Table Genes and corresponding conserved domains linked to integrative, conjugative and resistance elements found in contigs 5, and from the draft assembly of strain GP Families and E-values in bold indicate the best hits obtained with CONJscan [146] n.a., not applicable Reis et al BMC Genomics (2019) 20:885 Page of 23 Reis et al BMC Genomics (2019) 20:885 (MPF) system In this way, no complete type IV secretion systems were detected in contig suggesting this element may be mobile but possibly not conjugative Phylogenetic analysis As reported previously, strain GP shares the highest 16S rRNA gene sequence similarity with members of the genus Leucobacter, 94.6–96.9% (see Additional file Table S3), below the 98.7% threshold currently used to define a new species [26, 46] and close to the 97% threshold used to define a new genus [47] The phylogenetic analysis inferred from the alignment of the nearcomplete 16S rRNA gene between all fully sequenced Leucobacter spp showed that strain GP indeed clusters with Leucobacter spp (see Additional file Figure S3) Nevertheless, the ANI values between strain GP and the type strains of the validly named species of this genus ranged between 80.0 and 82.1% (Fig 3a, Additional file Table S3), well below the general species delimitation thresholds (94–96%) [48, 49], indicating that strain GP could not be affiliated to any of these species Average amino acid identity (AAI) comparisons between this strain and the type strains of the validly named species of this genus ranged between 64.2 and 69.1% (Fig 3b, Additional file Table S3) These values are near the lower edge of the typical genus delimitation boundaries (approximately 65%) [49], and the specific interspecies boundaries found between the analyzed type strains of Leucobacter spp (51.0–87.3%) This result was further supported by the percentage of conserved genes (POCP) [50] POCP values ranged between 46.7 and 56.5% (Fig 3c, Additional file Table S3), which is also on the lower edge of the interspecies boundaries found for this genus (42.0–81.3%) and the value suggested by Qin et al [50] for new genus delimitation (55%) The G + C content of strain GP was of 69.7% (Table 1), which, according to previous studies [51] is within the expected G + C interval (10%) for organisms of the same genus In fact, for the type strains of all validly named species of the Leucobacter genus, G + C content ranged between 64.5 and 71.0% (Table 1) Moreover, the phylogenetic analysis of 400 conserved proteins of Leucobacter spp using the PhyloPhlAn pipeline [52] revealed that although strain GP appears to share a common origin with the other isolates of Leucobacter spp (Fig 4), it also does not cluster with any of the analyzed strains Core and softcore genome of Leucobacter spp Orthologs gene cluster analysis with GET_HOMOLOGUES [54] revealed that Leucobacter spp and strain GP core and softcore genome contain 456 and 885 orthologs gene clusters, respectively (see Additional file Figure S4) However, only a fraction of these (approximately 50%) could be functionally annotated with eggNOG-Mapper and Page of 23 BlastKOALA [55, 56] This analysis revealed that most of these clusters are related to central metabolic pathways [57], including nucleotide and amino acid metabolism (118 clusters), and carbohydrate and lipid metabolism (16 clusters) (see Additional file Table S4), respectively Furthermore, these strains lack orthologs linked to antimicrobial resistance, quorum sensing, and biofilm formation, suggesting that they form a diverse and versatile genus with specific adaptations to different environments (see Additional file Table S4) Only a few of the fully sequenced Leucobacter spp analyzed are free-living organisms isolated from wastewater or soil These free-living strains did not form a clade The majority of the strains form facultative symbiotic associations with arthropods, nematodes, and plants (see Additional file Table S3) While Leucobacter sp AEAR [58], whose genome has been directly reconstructed from whole genome sequences of the nematodes Caenorhabditis angaria and Caenorhabditis remanei, could not be isolated, all Leucobacter spp symbionts were able to grow independently from their hosts Nevertheless, the analysis of strain’s AEAR genome revealed that it should be able to grow independently as all essential pathways seem to be present in its draft genome [58] This observation is further supported by the analysis of the genome of this strain (see Additional file Table S3) Unlike obligate symbionts, which often undergo extreme genome reduction [59–62], strain AEAR possesses a genome with similar size (3.54 Mb) and genetic density when compared to its closest relatives (Fig 4) Moreover, strain AEAR forms a monophyletic clade with Leucobacter sp Ag1 (accession no GCA_000980875.1) and other strains, which are all facultative symbionts from arthropod species able to grow independently from their hosts [63] These results suggest that the facultative living style may correlate with the phylogeny of the strains However, further studies are necessary in order to understand the link between phylogeny and lifestyle within this phylogenetic group Interestingly, strain GP appears to share many conserved genes with L chironomi DSM 19883T [64], a facultative symbiotic bacterium isolated from a member of the Chironomidae family (56.49% POCP, Fig 3c) Bidirectional best-hits (BDBH) analysis with GET_HOMOLOGUES of these two strains showed that they share 1372 orthologs gene clusters (data not shown), amounting to 38.6% of the total CDS of strain GP Most of these genes are linked to central metabolic pathways As strain GP, L chironomi also carries iron-heme acquisition operons hmuTUV (accessions no WP_024357741.1, WP_024357742.1 and WP_029747012.1, respectively) and efeUOB (accessions no WP_02436012.1, WP_ 024356011.1 and WP_024356010.1, respectively), and a homolog of heme oxygenase (hmuO, accession no WP_024356032.1) However, unlike in strain GP, L chironomi does not bear operons efeUOB and hmuTUV adjacently in its genome The efeUOB operon Reis et al BMC Genomics (2019) 20:885 Page of 23 Fig ANI (a), AAI (b) and POCP (c) heatmaps comparing values between strain GP and validly named species of the Leucobacter genus at the time of analysis Reis et al BMC Genomics (2019) 20:885 Page of 23 Fig Phylogenomic relationships between the Leucobacter genus and strain GP inferred from concatenated amino acid alignments of 400 universal proteins obtained with PhyloPhlAn [52] Representative members of genera Microbacterium, Leifsonia, Gulosibacter, Agromyces a n d Arthrobacter were included as outgroup Leucobacter spp strains sequenced in this study are marked with an asterisk, and sulfonamide degraders are shown in bold Node labels indicate local support values obtained with FastTree using the Shimodaira-Hasegawa test [53] The scale bar represents the number of expected substitutions per site The tree was rooted at the outgroup node and visualized with FigTree [125] and hmuO are absent from the softcore genome of Leucobacter spp., but are shared between several members of this genus (data not shown) Furthermore, strain GP also carries a chromate transport protein A (locus tag D3X82_06990) which was confirmed to be linked to chromate resistance and a common feature shared among several members of the Leucobacter genus [65] Estimation of gene loss in strain GP Prior studies suggested that strain GP was obligatorily dependent on A denitrificans PR1 for growth, as no isolated colonies of this organism were recovered after incubation in several conditions [26] Surprisingly, despite its dependent phenotype, strain GP did not show significant genome reduction as it is commonly reported in symbiotic bacteria [60] In fact, the number of genes and the genome size of this strain was similar to the ones found in other members of the Leucobacter genus (Table 2) These results suggest that, despite the PR1-dependent phenotype, strain GP differs from obligate parasites that, in the process of adapting to their hosts, undergo a process called reductive genome evolution, which results in relatively small genomes (often < Mb) [66, 67] (Table 3) Comparative genomic analysis of the Leucobacter genus revealed that the pangenome consists of 12,998 orthologous gene clusters The clusters present in at least 90% of the Leucobacter spp (28 of 31 genomes) were used as reference to determine potentially missing genes in the draft genome of strain GP These results were carefully analyzed and manually curated, due to the high frequency of annotation errors associated with draft assemblies [68, 69] In this way, of all Reis et al BMC Genomics (2019) 20:885 Page 10 of 23 Table Essential genes missing from the draft genome of strain GP identified by core/pangenome analysis with GET_HOMOLOGUES [54] Representative accession no L chironomi DSM 19883T Strain PR1 KO identifiers Description Pathway/System WP_024356349.1 ASC67664.1 K01476 Arginase RocF L-arginine biosynthesis; Urea cycle WP_024355584.1 Absent K08963, K08964 S-methyl-5-thioribose-1-phosphate isomerase MtnA Methionine salvage pathway WP_024357159.1 ASC65015.1 K06147, K06148, K16013, K16014 Thiol reductant ABC exporter subunit CydD Glutathione; L-cysteine ABC transporter WP_024357158.1 ASC65016.1 K06148, K16012 Thiol reductant ABC exporter subunit CydC WP_024356490.1 ASC65168.1 K02492 Glutamyl-tRNA reductase HemA WP_024356487.1 ASC64797.1 K01698 Porphobilinogen synthase HemB WP_084705356.1 ASC63016 K01749 Porphobilinogen deaminase HemC WP_024356489.1 ASC64317.1 K01599 Uroporphyrinogen decarboxylase HemE WP_024356124.1 ASC67862.1 K02083, K06016 Allantoate deiminase AllC these clusters, only 141 were present in 90% of the Leucobacter spp and were apparently absent from the draft genome of strain GP From these 141 clusters, only clusters were non hypothetical genes and no alternative pathways were found in the draft genome of strain GP (Table 3) Among these clusters, only those linked with tetrapyrrole biosynthesis (hemABCE) and thiol transporters (cydDC) may be linked to the incapacity of strain GP to grow independently, as both systems are essential for the synthesis and correct assembly of cytochromes [70–73] The possible absence of these regions from the genome of strain GP was further investigated by mapping the reads of its MAG against the genome of L chironomi DSM 19883T Visualization of the regions corresponding to these clusters on L chironomi (Table 3) further showed that no reads obtained from strain GP mapped to these regions (see Additional file Figure S5 and S6) The CydDC complex performs the transport of glutathione and L-cysteine and is responsible for maintaining an optimal redox balance in the periplasm [74, 75] This balance is crucial for the correct assembly of cytochromes in the plasma membrane, and its loss is usually associated with increased sensitivity to high temperature and oxidative stress [71–73] hemABCE encodes the proteins involved in the synthesis of tetrapyrroles and, subsequently, heme which acts as a prosthetic group in many respiratory and non-respiratory cytochromes [70] To the best of our knowledge, only a few bacterial strains have been found to be incapable of de novo heme biosynthesis [76] These strains are mainly pathogenic and affiliated to Haemophilus influenza, with the exception of the recently described environmental isolate Leucobacter sp strain ASN212 which requires exogenous heme for growth [76–78] These organisms rely on complex heme-acquisition systems to thrive in irondeficient environments and to synthesize essential hemecontaining proteins Functional analysis of the draft Porphyrin and chlorophyll metabolism Purine metabolism genome of strain GP revealed the presence of a heme ABC transport operon (hmuTUV) that encodes for a heminbinding periplasmic protein HmuT (locus tag D3X82_ 13650), a permease protein HmuU (D3X82_13655) and an ATP-binding protein HmuV (D3X82_13660), respectively This system has been extensively described and found to be highly conserved in the actinobacterium Corynebacterium diphtheria [76] However, in this organism, additional heme-binding genes (htaABC) and a heme oxygenase hmuO were found to be essential for successful heme and iron-heme acquisition [76] A homolog to hmuO was found in the genome of strain GP (locus tag D3X82_ 07630) However, the conserved htaABC operon, essential for exogenous heme-binding, appeared to be missing Instead of this operon, strain GP possesses a different adjacent gene cluster encoding for a deferrochelatase/ peroxidase EfeB (D3X82_13665), an iron uptake system component EfeO (D3X82_13670) and a ferrous iron permease EfeU (D3X82_13675) These enzymes have been previously linked to ferrous/ferric iron acquisition in Bacillus subtilis [79] and intact heme transport in Escherichia coli [80] However, to the best of our knowledge, the EfeUOB system has not been directly linked to intact heme-acquisition in Gram-positive bacteria In previous studies [26], we have supplied the consortium with exogenous heme and known heme precursors such as coproporphyrin III, coproporphyrin III tetramethylester and coproporphyrin I dihydrochloride, replicating the conditions that allowed the isolation of the heme-dependent Leucobacter sp ASN212 [26, 77] However, adding these metabolites to agar plates did not abolish the dependent phenotype of strain GP This result was unexpected as strain GP possesses several downstream genes of the porphyrin pathway; therefore, it should at least be able to use coproporphyrin III as a heme precursor This finding suggests that either the heme transport system of strain GP is ... differences in strain GP abundance are indicated by a, b, c and d (from higher to lower values of the mean) as determined by two-way ANOVA (pH, temperature and salinity) or one-way ANOVA (PR1 /GP ratio in. .. sequenced Leucobacter spp showed that strain GP indeed clusters with Leucobacter spp (see Additional file Figure S3) Nevertheless, the ANI values between strain GP and the type strains of the validly... regarding their respective tolerances toward temperature, pH and salinity (Fig 2) While the abundance of strain PR1 was constant when incubating at 22, 30 and 37 °C, respectively, strain GP abundance